It dipends on the properties of the protein, but the best methods is anyway the cromatography. First you better do an affinity cromatography and then a gel filtration.

Dear Priyanka,

if there was a King's way to solve this problem, there was no need for Biochemists :).

It really depends on what you start from and what you want to get and how you can detect your protein(s).

Usually, one will start with crude, but quick and cheap separation steps, capable of dealing with bulk amounts, like ammonium sulfate precipitation (fractionation) and bulk ion exchange, before going to more sophisticated methods that work only reasonably with smaller volumes and amounts.

However, as the very first step, I'd check the literature if there are already published procedures on the same protein or on similar proteins. Usually, you don't want to re-invent the wheel.

If you have a specific purification problem, let us know, we'll gladly try to assist!

There are many methods for protein separation that include ultra-centrifuge methods,solubility methods, immunological methods, column separation, chromatography and electrophoresis techniques etc. The method to be used depends on the protein you want to separate out. However , mostly column and chromatography methods are used.

HPLC

You have very interesting suggestions above. I will only add that is also important to take into account several factors that will condition the design of a protein purification strategy.

1/ What is the purpose of your protein purification? Is it analytical or preparative?

2/What type of assay do you have to follow your protein after each of the purification steps and whether or not it is important to preserve its native conformation? Most proteins are integral part of a complex. If using native conditions it might be difficult to break the interaction of your protein with the core complex.

3/ What is the percentage of purity to be satisfied at the end of your purification scheme? For example a protein estimated at 99% pure by SDS PAGE may well turn out not to be pure by other more sensitive techniques at the same time a protein with far less purity may well be suitable for MS/MS protein identification.

Of course there other factors I didn't mention.

Dear Priyanka,

Really its not very easy task to separate the protein of your interest out of the chunk. All the above suggestion are quite nice but need to see few things.

1. Is it an unknown protein or you know the amino acid composition and mass.

2. If you know the aa composition then you can actually predict the behavior of the protein and can use purification matrix accordingly ( or alternatively can get a synthetic gene too)

3. if u dont know the sequence, then you first need to do the fractionation using either TCA or ammonium sulphate ppt.

4. If you have any activity assay for your protein then you can go for FPLC and can broadly fractionate the multiple zones and then can check the activity. The fraction zone which has activity will help you in narrowing down the range or contaminating proteins.

Many more options are there but you need to proceed wisely.

A combination of various strategies would help isolate your protein of interest from a pool of many proteins.

1. Tabulate the properties of your protein such as its iso-electric point, His-tagged or not and so on.

2. Once you have all its properties listed, employ different purification techniques and find out how it works practically.

3. Combination or rearrangement of order of purification will help you optimize the purification protocol for your protein of interest.

The question is too wide and can be answered in many ways. A literature survey will be beneficial,

Best,

Sagar

U NEED TO STUDY THE PROTEIN CHARACTERISTICS SUCH AS CHARGE, SIZE, affinity to specific ligands of that protein. try to find purification methods for any other protein belonging to same family.

As most have mentioned, it depends on what you're purifying the protein from (recombinant from bacteria, from cell culture, etc.), whether you can add a tag for affinity purification, and how clean you need the protein. If you only need an enrichment and can tolerate some non-specific background proteins, 6xHis tags and Nickel resin work well and are cost effective. Although more expensive, FLAG tags or biotin acceptor peptides+strepavidin are good one-step methods to get a fairly clean prep. But I agree that more information about the protein, the starting material, and you're desired usage of the protein is required.

cheers,

Jordan